In spite of considerable progress in recent years towards the characterization of functional regions of HIV env proteins and in defining critical neutralizing epitopes, available vaccination strategies have not been effective in producing high titers of broadly crossreactive neutralizing antibodies. HIV env proteins are among the most highly glycosylated eucaryotic proteins, and recent studies have emphasized the importance of glycosylation in the formation of conformational structures necessary for proper function and immunogenicity of HIV env. We have recently isolated a chimpanzee monoclonal antibody, C108G, that has very potent neutralizing activity against some primary and laboratory strains of HIV-1. This antibody does not react with sites either in the V3 loop or the CD4-binding site, but recognizes a unique, carbohydrate-dependent epitope that we have localized to a site in or near the V2 loop of gpl20. Other investigators have isolated additional MAbs with similar properties that also map to the V2 domain. In the present proposal, we will use a novel glycopeptide expression system to express glycosylated fragments corresponding to both conserved and variable domains of HIV env in native forms. This expression system generates heterologous glycopeptide fragments linked to the N-terminal domain of the murine leukemia virus (MuLV) SU protein. The fused glycoproteins are highly expressed, efficiently secreted and easily purified by immunoaffinity methods. In contrast to previous studies, this system results in the production of properly folded and fully glycosylated domains, thereby allowing the characterization of conformational and carbohydrate-dependent epitopes. We will use this system to precisely map the epitopes recognized by C108G and other neutralizing MAbs directed against conformation and glycosylation dependent epitopes in the V1/V2 domains. We will also characterize the immunoreactivities and immunogenicities of different glycopeptide subregions of the V1/V2 domains and analyze the functional activities of antibodies directed against these regions that are present in sera of infected individuals and that are induced upon immunization with the purified fusion proteins. Similar methods will be used to express the V3 and V4/C4 domains of HIV-1 gpl20 in their correctly folded and glycosylated forms, and to evaluate their immunoreactivities and immunogenicities. These studies will be performed for a number of different viral strains, including primary and macrophage-tropic isolates. The goal of this work is to define native epitopes important for HIV neutralization and to identify additional domains capable of producing conserved neutralizing responses, that may have been missed by previous studies because of their conformational and glycan-dependent nature. Once such sites have been identified, novel features of our expression system can be used to produce these regions in highly immunogenic forms suitable as effective vaccines.